U.S. patent application number 11/734851 was filed with the patent office on 2008-01-03 for adenovirus fiber shaft composition and methods of use.
This patent application is currently assigned to GenVec, Inc.. Invention is credited to Cheng Cheng, Jason G. D. Gall, C. Richter King, Gary J. Nabel.
Application Number | 20080003236 11/734851 |
Document ID | / |
Family ID | 38876920 |
Filed Date | 2008-01-03 |
United States Patent
Application |
20080003236 |
Kind Code |
A1 |
King; C. Richter ; et
al. |
January 3, 2008 |
ADENOVIRUS FIBER SHAFT COMPOSITION AND METHODS OF USE
Abstract
The invention provides a gene transfer vector and a conjugate
comprising at least three contiguous amino acids of a shaft region
of a subgroup C adenovirus fiber protein. The invention also
provides methods of using the gene transfer vector and the
conjugate to induce an immune response in a mammal, and to deliver
a protein or a non-proteinaceous molecule to a specific cell
type.
Inventors: |
King; C. Richter;
(Washington, DC) ; Gall; Jason G. D.; (Germantown,
MD) ; Nabel; Gary J.; (Washington, DC) ;
Cheng; Cheng; (Bethesda, MD) |
Correspondence
Address: |
LEYDIG VOIT & MAYER, LTD
TWO PRUDENTIAL PLAZA, SUITE 4900
180 NORTH STETSON AVENUE
CHICAGO
IL
60601-6731
US
|
Assignee: |
GenVec, Inc.
Gaithersburg
MD
Government of the U.S.A., represented by the Secretary,
Department of Health and Human Services
Rockville
MD
|
Family ID: |
38876920 |
Appl. No.: |
11/734851 |
Filed: |
April 13, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US05/37155 |
Oct 17, 2005 |
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11734851 |
Apr 13, 2007 |
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60619912 |
Oct 18, 2004 |
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Current U.S.
Class: |
424/192.1 ;
424/196.11; 435/320.1; 435/69.1; 514/44R |
Current CPC
Class: |
Y02A 50/386 20180101;
Y02A 50/30 20180101; A61K 38/162 20130101; C12N 15/86 20130101;
C12N 2710/10322 20130101; C12N 2750/14143 20130101; Y02A 50/412
20180101; C12N 2710/10343 20130101; A61P 43/00 20180101; A61K
38/164 20130101 |
Class at
Publication: |
424/192.1 ;
424/196.11; 435/320.1; 435/069.1; 514/044 |
International
Class: |
A61K 38/00 20060101
A61K038/00; A61K 39/00 20060101 A61K039/00; A61K 39/385 20060101
A61K039/385; A61P 43/00 20060101 A61P043/00; C12N 15/00 20060101
C12N015/00; C12P 21/06 20060101 C12P021/06 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND
DEVELOPMENT
[0002] This invention was made in part with Government support
under Cooperative Research and Development Agreement (CRADA) Number
AI-1034, and amendments thereto, executed between GenVec, Inc. and
the U.S. Public Health Service representing the National Institute
of Allergy and Infectious Diseases. The Government may have certain
rights in this invention.
Claims
1. A method of inducing an immune response in a mammal, which
method comprises administering to the mammal a gene transfer vector
comprising (a) a nucleic acid sequence encoding at least one
antigen which is expressed in the mammal to induce an immune
response, and (b) an amino acid sequence comprising at least three
contiguous amino acids of a shaft region of a subgroup C adenovirus
fiber protein, wherein the gene transfer vector is not an
adenoviral vector.
2. The method of claim 1, wherein the gene transfer vector is a
viral vector.
3. The method of claim 2, wherein the viral vector is selected from
the group consisting of adeno-associated virus vectors, retroviral
vectors, herpes simplex virus (HSV) vectors, poxvirus vectors,
lentiviral vectors, parvovirus vectors, and bacteriophage
vectors.
4. The method of claim 1, wherein the gene transfer vector is a
liposome, a plasmid, or a virus-like particle.
5. A method of inducing an immune response in a mammal, which
method comprises administering to the mammal a conjugate comprising
(a) at least one antigen which induces an immune response in the
mammal, and (b) an amino acid sequence comprising at least three
contiguous amino acids of a shaft region of a subgroup C adenovirus
fiber protein, wherein the conjugate is not an adenovirus.
6. The method of claim 5, wherein the conjugate is a fusion
protein.
7. The method of claim 1, wherein the amino acid sequence comprises
at least three contiguous amino acids of a shaft region of a
serotype 5 or serotype 2 adenovirus fiber protein.
8. The method of claim 1, wherein the amino acid sequence comprises
the amino acid residues lysine-lysine-threonine-lysine (KKTK) (SEQ
ID NO: 1).
9. The method of claim 1, wherein the antigen is selected from the
group consisting of a peptide, a protein, or a glycoprotein.
10. The method of claim 1, wherein at least one antigen is selected
from the group consisting of env, gag, and pol from clades A, B, or
C of a human immunodeficiency virus (HIV), and a fusion protein
comprising any of the foregoing.
11. The method of claim 1, wherein at least one antigen is selected
from the group consisting of an E protein, an M protein, and a
spike protein of a severe acute respiratory syndrome (SARS)
virus.
12. The method of claim 1, wherein at least one antigen is isolated
from Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, or
Plasmodium malariae.
13. The method of claim 1, wherein at least one antigen is selected
from the group consisting of a Dengue protein M, Dengue protein E,
Dengue D1NS1, Dengue D1NS2, and Dengue D1NS3.
14. The method of claim 1, wherein the mammal is a human.
15. A gene transfer vector comprising (a) a nucleic acid sequence
encoding a protein, and (b) an amino acid sequence comprising at
least three contiguous amino acids of a shaft region of a subgroup
C adenovirus fiber protein, wherein the gene transfer vector is not
an adenoviral vector.
16. The gene transfer vector of claim 15, wherein the nucleic acid
sequence encodes an antigen or a cytotoxic protein.
17. The gene transfer vector of claim 15, wherein the gene transfer
vector is a viral vector.
18. The gene transfer vector of claim 17, wherein the viral vector
is selected from the group consisting of adeno-associated virus
vectors, retroviral vectors, herpes simplex virus (HSV) vectors,
poxvirus vectors, lentiviral vectors, parvovirus vectors, and
bacteriophage vectors.
19. The gene transfer vector of claim 15, wherein the gene transfer
vector is a liposome, a plasmid, or a virus-like particle.
20. The gene transfer vector of claim 15, wherein the amino acid
sequence comprises at least three contiguous amino acids of a shaft
region of a serotype 2 or serotype 5 adenovirus fiber protein.
21. The gene transfer vector of claim 15, wherein the amino acid
sequence comprises the amino acid residues
lysine-lysine-threonine-lysine (KKTK) (SEQ ID NO: 1).
22. A conjugate comprising (a) a protein or a non-proteinaceous
molecule, and (b) an amino acid sequence comprising at least three
contiguous amino acids of a shaft region of a subgroup C adenovirus
fiber protein, wherein when the conjugate comprises a protein, the
conjugate is not an adenovirus.
23. The conjugate of claim 22, wherein the conjugate comprises a
protein.
24. The conjugate of claim 23, wherein the protein is an antigen or
a cytotoxic protein.
25. The conjugate of claim 22, wherein the conjugate is a fusion
protein.
26. The conjugate of claim 22, wherein the conjugate comprises a
non-proteinaceous molecule.
27. The conjugate of claim 26, wherein the non-proteinaceous
material is a small molecule.
28. The conjugate of claim 27, wherein the small molecule is a
hapten.
29. The conjugate of claim 22, wherein the amino acid sequence
comprises at least three contiguous amino acids of a shaft region
of a serotype 2 or serotype 5 adenovirus fiber protein.
30. The conjugate of claim 22, wherein the amino acid sequence
comprises the amino acid residues lysine-lysine-threonine-lysine
(KKTK) (SEQ ID NO: 1).
31. A method of delivering a protein to a cell, which method
comprises contacting the cell with the gene transfer vector of
claim 15, whereby the nucleic acid sequence is expressed and the
protein is produced.
32. A method of delivering a protein to a cell, which method
comprises contacting the cell with the conjugate of claim 23,
whereby the nucleic acid sequence is expressed and the protein is
produced.
33. A method of delivering a non-proteinaceous molecule to a cell,
which method comprises contacting the cell with the conjugate of
claim 26.
34. The method of claim 31, wherein the cell is a dendritic
cell.
35. The method of claim 31, wherein the cell is a tumor cell.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation of copending
International Patent Application No. PCT/US2005/037155, filed Oct.
17, 2005, which claims the benefit of U.S. Provisional Patent
Application No. 60/619,912, filed Oct. 18, 2004.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
[0003] Incorporated by reference in its entirety herein is a
computer-readable nucleotide/amino acid sequence listing submitted
concurrently herewith and identified as follows: One 823 Byte ASCII
(Text) file named "701534_ST25.txt," created on Apr. 7, 2007.
FIELD OF THE INVENTION
[0004] This invention pertains to compositions comprising a portion
of an adenovirus fiber protein, and methods of using same.
BACKGROUND OF THE INVENTION
[0005] Delivery of proteins as therapeutics or for inducing an
immune response in biologically relevant amounts has been an
obstacle to drug and vaccine development for decades. One solution
that has proven to be a successful alternative to traditional drug
delivery approaches is delivery of exogenous nucleic acid sequences
for production of therapeutic factors in vivo. Gene transfer
vectors ideally enter a wide variety of cell types, have the
capacity to accept large nucleic acid sequences, are safe, and can
be produced in quantities required for treating patients. Viral
vectors have these advantageous properties and are used in a
variety of protocols to treat or prevent biological disorders.
[0006] Despite their advantageous properties, widespread use of
viral gene transfer vectors is hindered by several factors. In this
regard, certain cells are not readily amenable to gene delivery by
currently available viral vectors. For example, lymphocytes are
impaired in the uptake of adenoviruses (Silver et al., Virology
165, 377-387 (1988); Horvath et al., J. Virology, 62(1), 341-345
(1988)).
[0007] The use of viral gene transfer vectors also is impeded by
the immunogenicity of viral vectors. A majority of the U.S.
population has been exposed to wild-type forms of many of the
viruses currently under development as gene transfer vectors (e.g.,
adenovirus). As a result, much of the U.S. population has developed
pre-existing immunity to certain virus-based gene transfer vectors.
As a result, such vectors are quickly cleared from the bloodstream,
thereby reducing the effectiveness of the vector in delivering
biologically relevant amounts of a gene product. Moreover, the
immunogenicity of certain viral vectors prevents efficient repeat
dosing, which can be advantageous for "boosting" the immune system
against pathogens, and results in only a small fraction of a dose
of the viral vector delivering its payload to host cells.
[0008] Thus, there remains a need for improved methods of
delivering therapeutic or antigenic genes and proteins to target
cells. The invention provides such a method. These and other
advantages of the invention, as well as additional inventive
features, will be apparent from the description of the invention
provided herein.
BRIEF SUMMARY OF THE INVENTION
[0009] The invention provides a method of inducing an immune
response in a mammal comprising administering to the mammal a gene
transfer vector comprising (a) a nucleic acid sequence encoding at
least one antigen which is expressed in the mammal to induce an
immune response, and (b) an amino acid sequence comprising at least
three contiguous amino acids of a shaft region of a subgroup C
adenovirus fiber protein, wherein the gene transfer vector is not
an adenoviral vector.
[0010] The invention also provides a method of inducing an immune
response in a mammal comprising administering to the mammal a
conjugate comprising (a) at least one antigen which induces an
immune response in the mammal, and (b) an amino acid sequence
comprising at least three contiguous amino acids of a shaft region
of a subgroup C adenovirus fiber protein, wherein the conjugate is
not an adenovirus.
[0011] The invention additionally provides a gene transfer vector
comprising (a) a nucleic acid sequence encoding a protein, and (b)
an amino acid sequence comprising at least three contiguous amino
acids of a shaft region of a subgroup C adenovirus fiber protein,
wherein the gene transfer vector is not an adenoviral vector.
[0012] The invention further provides a conjugate comprising (a) a
protein or a non-proteinaceous molecule, and (b) an amino acid
sequence comprising at least three contiguous amino acids of a
shaft region of a subgroup C adenovirus fiber protein, wherein when
the conjugate comprises a protein, the conjugate is not an
adenovirus.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1A is a graph illustrating the CD4+ T cell response in
mice administered with adenoviral vector constructs encoding a
green fluorescent protein (GFP).
[0014] FIG. 1B is a graph illustrating the CD8+ T cell response in
mice administered with adenoviral vector constructs encoding a
green fluorescent protein.
[0015] FIG. 2 is a graph illustrating luciferase expression
following administration of the adenoviral vectors AdlucDA, Adluc,
and Adf to murine bone marrow dendritic cells in the presence or
absence of a glycosaminoglycan competitor.
[0016] FIGS. 3A-3D are each a graph illustrating luciferase
expression following administration of the adenoviral vectors AdL
and AdDA to 293-ORF6 cells, Caov3 human ovarian adenocarcinoma
cells, LL2 murine lung carcinoma cells, and CT26 murine colon
carcinoma cells, respectively.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The invention is predicated, at least in part, on the
discovery that a subgroup C adenoviral vector ablated for native
host cell binding is capable of inducing an immune response in a
human host when the adenoviral vector encodes an antigenic
transgene. Such an adenoviral vector typically comprises a fiber
protein in which the knob domain of the fiber is altered or
deleted, but retains the shaft region of the fiber.
[0018] The invention provides a method of inducing an immune
response in a mammal, which method comprises administering to the
mammal a gene transfer vector comprising (a) a nucleic acid
sequence encoding at least one antigen which is expressed in the
mammal to induce an immune response, and (b) an amino acid sequence
comprising at least three contiguous amino acids of a shaft region
of a subgroup C adenovirus fiber protein, wherein the gene transfer
vector is not an adenoviral vector.
[0019] The gene transfer vector can be any suitable gene transfer
vector. Examples of suitable gene transfer vectors include
plasmids, liposomes, molecular conjugates (e.g., transferrin), and
viruses. Preferably, the gene transfer vector is a viral vector.
Any suitable viral vector can be used in the inventive method, so
long as the viral vector is not an adenoviral vector. Suitable
viral vectors include, for example, retroviral vectors, herpes
simplex virus (HSV)-based vectors, poxvirus vectors, lentivirus
vectors, parvovirus-based vectors, e.g., adeno-associated virus
(AAV)-based vectors, and bacteriophage vectors. These viral vectors
can be prepared using standard recombinant DNA techniques described
in, for example, Sambrook et al., Molecular Cloning, A Laboratory
Manual, 3.sup.rd edition, Cold Spring Harbor Press, Cold Spring
Harbor, N.Y. (2001), and Ausubel et al., Current Protocols in
Molecular Biology, Greene Publishing Associates and John Wiley
& Sons, New York, N.Y. (1994).
[0020] Retrovirus is an RNA virus capable of infecting a wide
variety of host cells. Upon infection, the retroviral genome
integrates into the genome of its host cell and is replicated along
with host cell DNA, thereby constantly producing viral RNA and any
nucleic acid sequence incorporated into the retroviral genome. As
such, long-term expression of a therapeutic factor(s) is achievable
when using retrovirus. Retroviruses contemplated for use in gene
therapy are relatively non-pathogenic, although pathogenic
retroviruses exist. When employing pathogenic retroviruses, e.g.,
human immunodeficiency virus (HIV) or human T-cell lymphotrophic
viruses (HTLV), care must be taken in altering the viral genome to
eliminate toxicity to the host. A retroviral vector additionally
can be manipulated to render the virus replication-deficient. As
such, retroviral vectors are considered particularly useful for
stable gene transfer in vivo. Lentiviral vectors, such as HIV-based
vectors, are exemplary of retroviral vectors used for gene
delivery. Unlike other retroviruses, HIV-based vectors are known to
incorporate their passenger genes into non-dividing cells and,
therefore, can be of use in treating persistent forms of
disease.
[0021] An HSV-based viral vector is suitable for use as a gene
transfer vector to introduce a nucleic acid into numerous cell
types. The mature HSV virion consists of an enveloped icosahedral
capsid with a viral genome consisting of a linear double-stranded
DNA molecule that is 152 kb. Most replication-deficient HSV vectors
contain a deletion to remove one or more intermediate-early genes
to prevent replication. Advantages of the HSV vector are its
ability to enter a latent stage that can result in long-term DNA
expression, and its large viral DNA genome that can accommodate
exogenous DNA inserts of up to 25 kb. Of course, the ability of HSV
to promote long-term production of exogenous protein is potentially
disadvantageous in terms of short-term treatment regimens. However,
one of ordinary skill in the art has the requisite understanding to
determine the appropriate vector for a particular situation.
HSV-based vectors are described in, for example, U.S. Pat. Nos.
5,837,532, 5,846,782, 5,849,572, and 5,804,413, and International
Patent Application Publication Nos. WO 91/02788, WO 96/04394, WO
98/15637, and WO 99/06583.
[0022] Because they are highly immunogenic and readily engineered,
poxvirus vectors have been used extensively as vaccines against
infectious organisms and, more recently, as tumor vaccines.
Vaccinia virus and other members of the poxviridae family remain in
the cytoplasm and use virally encoded polymerases to carry out
replication and transcription. Thus, recombination of viral DNA
into the genome is not a concern with vaccinia virus, as it is with
other vectors, particularly retroviruses. The infectious cycle is
divided into three phases. Early-phase genes, typically encoding
proteins with enzymatic function, are expressed before replication.
The expression of a small number of intermediate genes depends on
replication of the genome and, in turn, drives expression of
structural proteins and other products of the late genes.
[0023] AAV vectors are viral vectors of particular interest for use
in vaccine protocols. AAV is a DNA virus, which is not known to
cause human disease. The AAV genome is comprised of two genes, rep
and cap, flanked by inverted terminal repeats (ITRs), which contain
recognition signals for DNA replication and packaging of the virus.
AAV requires co-infection with a helper virus (i.e., an adenovirus
or a herpes simplex virus), or expression of helper genes, for
efficient replication. AAV can be propagated in a wide array of
host cells including human, simian, and rodent cells, depending on
the helper virus employed. An AAV vector used for administration of
a nucleic acid sequence typically has approximately 96% of the
parental genome deleted, such that only the ITRs remain. The use of
AAV as a vaccine construct against HIV is reviewed in, for example,
Expert. Rev. Vaccines, 1, 7 (2002).
[0024] Bacteriophage (or phage) are viruses that grow in bacterial
cells. Depending on the complexity of the bacteriophage genome,
some bacteriophage rely entirely upon the protein machinery of the
host cell for propagation. Bacteriophage propagate by way of a
lytic or lysogenic life cycle. A bacteriophage in the lytic cycle
converts an infected cell into a "phage factory," and produces many
phage progeny. Bacteriophage capable only of lytic growth often are
referred to as a "virulent" bacteriophage. The lysogenic life cycle
has been observed only with bacteriophage containing a
double-strand DNA genome, and typically involves the bacteriophage
integrating into the bacterial chromosome. No progeny bacteriophage
particles are produced during the lysogenic cycle. Bacteriophage
capable of a lysogenic life cycle are often referred to as
"temperate" bacteriophage, and can undergo a lytic cycle under
certain conditions, such as DNA damage (see, e.g., Maloy et al.,
eds., Microbial Genetics, 2.sup.nd ed., Jones and Bartlett
Publishers, Boston (1994)). Bacteriophage have been modified to
specifically target and transduce mammalian cells, (see, e.g., Poul
et al., J. Mol. Biol., 288, 203-11 (1999), Monaci et al., Curr.
Opin. Mol. Ther., 3, 159-69 (2001), and Larocca et al., Curr.
Pharm. Biotechnol., 3, 45-57 (2002)). Bacteriophage suitable for
use as gene transfer vectors include, for example, bacteriophage
.lamda. and bacteriophage M13. The use of bacteriophage as a human
vaccine vector is reviewed in, for example, Clark et al., Expert.
Rev. Vaccines, 3, 463-76 (2004).
[0025] A virus-like particle is a defective virion which is
incapable of infecting a host cell due to the presence of one or
more genetic modifications of viral genes or other genetic elements
which are functionally critical at some stage of the virus
lifecycle. Virus-like particles may or may not contain all of the
viral proteins normally found in infectious virions and may or may
not contain nucleic acid (i.e., DNA or RNA). If nucleic acid is
contained within the particle, it will be incapable of infecting a
host cell. Examples of virus like particle vaccine constructs are
disclosed in, for example, Schreckenberger et al., Curr. Opin.
Oncol., 16, 485-91 (2004).
[0026] The gene transfer vector of the inventive method comprises a
nucleic acid sequence encoding an antigen which is expressed in the
mammal to induce an immune response. An "antigen" is a molecule
that triggers an immune response in a mammal. An "immune response"
can entail, for example, antibody production and/or the activation
of immune effector cells. An antigen in the context of the
invention can comprise any subunit of any proteinaceous molecule,
including a protein, peptide, or glycoprotein of viral, bacterial,
parasitic, fungal, protozoan, prion, cellular, or extracellular
origin, which ideally provokes an immune response in mammal,
preferably leading to protective immunity. The antigen also can be
a self antigen, i.e., an autologous protein which the body reacts
to as if it is a foreign invader. The nucleic acid sequence
encoding the antigen is not limited to a type of nucleic acid
sequence or any particular origin. For example, the nucleic acid
sequence can be recombinant DNA, can be genomic DNA, can be
obtained from a DNA library of potential antigenic epitopes, or can
be synthetically generated.
[0027] In one embodiment, the antigen is a viral antigen. The viral
antigen can be isolated from any virus including, but not limited
to, a virus from any of the following viral families: Arenaviridae,
Arterivirus, Astroviridae, Baculoviridae, Badnavirus, Barnaviridae,
Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae,
Capillovirus, Carlavirus, Caulimovirus, Circoviridae,
Closterovirus, Comoviridae, Coronaviridae (e.g., Coronavirus, such
as severe acute respiratory syndrome (SARS) virus), Corticoviridae,
Cystoviridae, Deltavirus, Dianthovirus, Enamovirus, Filoviridae
(e.g., Marburg virus and Ebola virus (e.g., Zaire, Reston, Ivory
Coast, or Sudan strain)), Flaviviridae, (e.g., Hepatitis C virus,
Dengue virus 1, Dengue virus 2, Dengue virus 3, and Dengue virus
4), Hepadnaviridae (e.g., Hepatitis B virus), Herpesviridae (e.g.,
Human herpesvirus 1, 3, 4, 5, and 6, and Cytomegalovirus),
Hypoviridae, Iridoviridae, Leviviridae, Lipothrixviridae,
Microviridae, Orthomyxoviridae (e.g., Influenzavirus A and B),
Papovaviridae, Paramyxoviridae (e.g., measles, mumps, and human
respiratory syncytial virus), Parvoviridae, Picornaviridae (e.g.,
enterovirus, poliovirus, rhinovirus, hepatovirus, and aphthovirus),
Plasmodiidae (e.g., Plasmodium falciparum, Plasmodium vivax,
Plasmodium ovale, and Plasmodium malariae), Poxviridae (e.g.,
vaccinia virus), Reoviridae (e.g., rotavirus), Retroviridae (e.g.,
lentivirus, such as human immunodeficiency virus (HIV) 1 and HIV
2), Rhabdoviridae, and Totiviridae. Preferably, at least one
antigen of the inventive method is a retroviral antigen. The
retroviral antigen can be, for example, an HIV antigen, such as all
or part of the gag, env, or pol protein. Any clade of HIV is
appropriate for antigen selection, including clades A, B, C, MN,
and the like. Also preferably, at least one antigen encoded by the
gene transfer vector is a coronavirus antigen, such as a SARS virus
antigen. Suitable SARS virus antigens for the inventive method
include, for example, all or part of the E protein, the M protein,
and the spike protein of the SARS virus. Suitable viral antigens
also include all or part of Dengue protein M, Dengue protein E,
Dengue D1NS1, Dengue D1NS2, and Dengue D1NS3. The antigenic
peptides specifically recited herein are merely exemplary as any
viral protein can be used in the context of the invention.
[0028] The antigen can be a parasite antigen such as, but not
limited to, a Sporozoan antigen. For example, the nucleic acid
sequence can encode a Plasmodian antigen, such as all or part of a
Circumsporozoite protein, a Sporozoite surface protein, a liver
stage antigen, an apical membrane associated protein, or a
Merozoite surface protein.
[0029] Alternatively or in addition, at least one antigen encoded
by the viral vector is a bacterial antigen. The antigen can
originate from any bacterium including, but not limited to,
Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio,
Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium,
Cytophaga, Deinococcus, Escherichia, Halobacterium, Heliobacter,
Hyphomicrobium, Methanobacterium, Micrococcus, Myobacterium,
Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria,
Prochloron, Proteus, Pseudomonas, Phodospirillum, Rickettsia,
Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus,
Streptococcus, Streptomyces, Sulfolobus, Thermoplasma,
Thiobacillus, and Treponema. In a preferred embodiment, at least
one antigen encoded by the nucleic acid sequence is a Pseudomonas
antigen or a Heliobacter antigen.
[0030] It will be appreciated that an entire, intact viral or
bacterial protein is not required to produce an immune response.
Indeed, most antigenic epitopes are relatively small in size and,
therefore, protein fragments can be sufficient for exposure to the
immune system of the mammal. In addition, a fusion protein can be
generated between two or more antigenic epitopes of one or more
antigens. For example, all or part of HIV envelope, e.g., all or
part of gp120 or gp160, can be fused to all or part of the HIV pol
protein to generate a more complete immune response against the HIV
pathogen compared to that generated by a single epitope. Delivery
of fusion proteins via a gene transfer vector to a mammal allows
exposure of an immune system to multiple antigens and, accordingly,
enables a single vaccine composition to provide immunity against
multiple pathogens or multiple epitopes of a single pathogen.
[0031] Preferably, the nucleic acid is operably linked to (i.e.,
under the transcriptional control of) one or more promoter and/or
enhancer elements, for example, as part of a promoter-variable
expression cassette. Techniques for operably linking sequences
together are well known in the art. A "promoter" is a DNA sequence
that directs the binding of RNA polymerase and thereby promotes RNA
synthesis. A nucleic acid sequence is "operably linked" to a
promoter when the promoter is capable of directing transcription of
that nucleic acid sequence. A promoter can be native or non-native
to the nucleic acid sequence to which it is operably linked.
[0032] Any promoter (i.e., whether isolated from nature or produced
by recombinant DNA or synthetic techniques) can be used in
connection with the invention to provide for transcription of the
nucleic acid sequence. The promoter preferably is capable of
directing transcription in a eukaryotic (desirably mammalian) cell.
The functioning of the promoter can be altered by the presence of
one or more enhancers and/or silencers present on the vector.
"Enhancers" are cis-acting elements of DNA that stimulate or
inhibit transcription of adjacent genes. An enhancer that inhibits
transcription also is termed a "silencer." Enhancers differ from
DNA-binding sites for sequence-specific DNA binding proteins found
only in the promoter (which also are termed "promoter elements") in
that enhancers can function in either orientation, and over
distances of up to several kilobase pairs (kb), even from a
position downstream of a transcribed region.
[0033] Promoter regions can vary in length and sequence and can
further encompass one or more DNA binding sites for
sequence-specific DNA binding proteins and/or an enhancer or
silencer. Enhancers and/or silencers can similarly be present on a
nucleic acid sequence outside of the promoter per se. Desirably, a
cellular or viral enhancer, such as the cytomegalovirus (CMV)
immediate-early enhancer, is positioned in the proximity of the
promoter to enhance promoter activity.
[0034] Any suitable promoter or enhancer sequence can be used in
the context of the invention. In this respect, the antigen-encoding
nucleic acid sequence can be operably linked to a viral promoter.
Suitable viral promoters include, for instance, cytomegalovirus
(CMV) promoters, such as the CMV immediate-early promoter
(described in, for example, U.S. Pat. Nos. 5,168,062 and
5,385,839), promoters derived from human immunodeficiency virus
(HIV), such as the HIV long terminal repeat promoter, Rous sarcoma
virus (RSV) promoters, such as the RSV long terminal repeat, mouse
mammary tumor virus (MMTV) promoters, HSV promoters, such as the
Lap2 promoter or the herpes thymidine kinase promoter (Wagner et
al., Proc. Natl. Acad. Sci., 78, 144-145 (1981)), promoters derived
from SV40 or Epstein Barr virus, an adeno-associated viral
promoter, such as the p5 promoter, and the like.
[0035] Alternatively, the invention employs a cellular promoter,
i.e., a promoter that drives expression of a cellular protein.
Preferred cellular promoters for use in the invention will depend
on the desired expression profile to produce the antigen(s). In one
aspect, the cellular promoter is preferably a constitutive promoter
that works in a variety of cell types, such as immune cells
described herein. Suitable constitutive promoters can drive
expression of genes encoding transcription factors, housekeeping
genes, or structural genes common to eukaryotic cells. For example,
the Ying Yang 1 (YY1) transcription factor (also referred to as
NMP-1, NF-E1, and UCRBP) is a ubiquitous nuclear transcription
factor that is an intrinsic component of the nuclear matrix (Guo et
al., PNAS, 92, 10526-10530 (1995)). While these promoters are
considered constitutive promoters, it is understood in the art that
constitutive promoters can be upregulated. Promoter analysis shows
that the elements critical for basal transcription reside from -277
to +475 of the YY1 gene relative to the transcription start site
from the promoter, and include a TATA and CCAAT box. JEM-1 (also
known as HGMW and BLZF-1) also is a ubiquitous nuclear
transcription factor identified in normal and tumor tissues (Tong
et al., Leukemia, 12(11), 1733-1740 (1998), and Tong et al.,
Genomics, 69(3), 380-390 (2000)). JEM-1 is involved in cellular
growth control and maturation, and can be upregulated by retinoic
acids. Sequences responsible for maximal activity of the JEM-1
promoter have been located at -432 to +101 of the JEM-1 gene
relative the transcription start site of the promoter. Unlike the
YY1 promoter, the JEM-1 promoter does not comprise a TATA box. The
ubiquitin promoter, specifically UbC, is a strong constitutively
active promoter functional in several species. The UbC promoter is
further characterized in Marinovic et al., J. Biol. Chem., 277(19),
16673-16681 (2002).
[0036] The promoter also can be a regulatable promoter, i.e., a
promoter that is up- and/or down-regulated in response to
appropriate signals. The use of a regulatable promoter or
expression control sequence is particularly applicable to DNA
vaccine development as antigenic proteins, including viral and
parasite antigens, frequently are toxic to cell lines used to
produce the gene transfer vector. In one embodiment, the regulatory
sequences operably linked to the antigen-encoding nucleic acid
sequence include components of the tetracycline expression system,
e.g., tet operator sites. For instance, the antigen-encoding
nucleic acid sequence is operably linked to a promoter which is
operably linked to one or more tet operator sites. A gene transfer
vector comprising such an expression cassette can be propagated in
a cell line which comprises a nucleic acid sequence encoding a tet
repressor protein. By producing the tet repressor protein in the
cell line, antigen production is inhibited and propagation proceeds
without any associated antigen-mediated toxicity. Suitable
regulatable promoter systems also include, but are not limited to,
the IL-8 promoter, the metallothionine inducible promoter system,
the bacterial lacZYA expression system, and the T7 polymerase
system. Further, promoters that are selectively activated at
different developmental stages (e.g., globin genes are
differentially transcribed from globin-associated promoters in
embryos and adults) can be employed. The promoter sequence can
contain at least one regulatory sequence responsive to regulation
by an exogenous agent. The regulatory sequences are preferably
responsive to exogenous agents such as, but not limited to, drugs,
hormones, radiation, or other gene products.
[0037] The promoter can be a tissue-specific promoter, i.e., a
promoter that is preferentially activated in a given tissue and
results in expression of a gene product in the tissue where
activated. A tissue-specific promoter suitable for use in the
invention can be chosen by the ordinarily skilled artisan based
upon the target tissue or cell-type. Preferred tissue-specific
promoters for use in the inventive method are specific to immune
cells, such as the dendritic-cell specific Dectin-2 promoter
described in Morita et al., Gene Ther., 8, 1729-37 (2001).
[0038] In yet another embodiment, the promoter can be a chimeric
promoter. A promoter is "chimeric" in that it comprises at least
two nucleic acid sequence portions obtained from, derived from, or
based upon at least two different sources (e.g., two different
regions of an organism's genome, two different organisms, or an
organism combined with a synthetic sequence). Preferably, the two
different nucleic acid sequence portions exhibit less than about
40%, more preferably less than about 25%, and even more preferably
less than about 10% nucleic acid sequence identity to one another
(which can be determined by methods described elsewhere herein).
Any suitable chimeric promoter can be used in the inventive method.
Preferably, the chimeric promoter is comprised of a functional
portion of a viral promoter and a functional portion of a cellular
promoter. More preferably, the chimeric promoter comprises a
functional portion of a viral promoter and a functional portion of
a cellular promoter that is radiation-inducible. Most preferably,
the chimeric promoter comprises a functional portion of a CMV
promoter and a functional portion of an EGR-1 promoter (i.e., a
chimeric "CMV/EGR-1" promoter). The functional portion of the CMV
promoter preferably is derived from a human CMV, and more
particularly from the human CMV immediate early (IE)
promoter/enhancer region (see, e.g., U.S. Pat. Nos. 5,168,062 and
5,385,839). In addition, the functional portion of the EGR-1
promoter preferably comprises one or more CArG domains of an EGR-1
promoter, as described in, for example, U.S. Pat. Nos. 6,579,522
and 6,605,712. In a particularly preferred embodiment of the
invention, the chimeric promoter comprises a functional portion of
the CMV IE enhancer/promoter region, and an EGR-1 promoter
comprising six CArG domains. In this manner, the portion of the CMV
IE enhancer/promoter region functions as an enhancer for the EGR-1
promoter. Chimeric promoters can be generated using standard
molecular biology techniques, such as those described in Sambrook
et al., supra, and Ausubel et al., supra.
[0039] A "functional portion" of a promoter is any portion of a
promoter that measurably promotes, enhances, or controls expression
(typically transcription) of an operatively linked nucleic acid.
Such regulation of expression can be measured via RNA or protein
detection by any suitable technique, and several such techniques
are known in the art. Examples of such techniques include Northern
analysis (see, e.g., Sambrook et al., supra, and McMaster and
Carmichael, PNAS, 74, 4835-4838 (1977)), RT-PCR (see, e.g., U.S.
Pat. No. 5,601,820, and Zaheer et al., Neurochem Res., 20,
1457-1463 (1995)), in situ hybridization methods (see, e.g., U.S.
Pat. Nos. 5,750,340 and 5,506,098), antibody-mediated techniques
(see, e.g., U.S. Pat. Nos. 4,376,110, 4,452,901, and 6,054,467),
and promoter assays utilizing reporter gene systems such as the
luciferase gene (see, e.g., Taira et al., Gene, 263, 285-292
(2001)). Eukaryotic expression systems in general are further
described in Sambrook et al., supra.
[0040] A promoter can be selected for use in the method of the
invention by matching its particular pattern of activity with the
desired pattern and level of expression of the antigen(s). For
example, the gene transfer vector can comprise two or more nucleic
acid sequences that encode different antigens and are operably
linked to different promoters displaying distinct expression
profiles. For example, a first promoter is selected to mediate an
initial peak of antigen production, thereby priming the immune
system against an encoded antigen. A second promoter is selected to
drive production of the same or different antigen such that
expression peaks several days after that of the first promoter,
thereby "boosting" the immune system against the antigen.
Alternatively, a chimeric promoter can be constructed which
combines the desirable aspects of multiple promoters. For example,
a CMV-RSV hybrid promoter combining the CMV promoter's initial rush
of activity with the RSV promoter's high maintenance level of
activity is especially preferred for use in many embodiments of the
inventive method. In that antigens can be toxic to eukaryotic
cells, it may be advantageous to modify the promoter to decrease
activity in cell lines used to propagate the gene transfer
vector.
[0041] To optimize protein production, preferably the nucleic acid
sequence encoding the antigen further comprises a polyadenylation
site following the coding sequence of the antigen-encoding nucleic
acid sequence. Any suitable polyadenylation sequence can be used,
including a synthetic optimized sequence, as well as the
polyadenylation sequence of BGH (Bovine Growth Hormone), polyoma
virus, TK (Thymidine Kinase), EBV (Epstein Barr Virus), and the
papillomaviruses, including human papillomaviruses and BPV (Bovine
Papilloma Virus). A preferred polyadenylation sequence is the SV40
(Human Sarcoma Virus-40) polyadenylation sequence. Also, preferably
all the proper transcription signals (and translation signals,
where appropriate) are correctly arranged such that the nucleic
acid sequence is properly expressed in the cells into which it is
introduced. If desired, the nucleic acid sequence also can
incorporate splice sites (i.e., splice acceptor and splice donor
sites) to facilitate mRNA production.
[0042] If the antigen-encoding nucleic acid sequence encodes a
processed or secreted protein or peptide, or a protein that acts
intracellularly, preferably the antigen-encoding nucleic acid
sequence further comprises the appropriate sequences for
processing, secretion, intracellular localization, and the like.
The antigen-encoding nucleic acid sequence can be operably linked
to a signal sequence, which targets a protein to cellular machinery
for secretion. Appropriate signal sequences include, but are not
limited to, leader sequences for immunoglobulin heavy chains and
cytokines, (see, e.g., Ladunga, Current Opinions in Biotechnology,
11, 13-18 (2000)). Other protein modifications can be required to
secrete a protein from a host cell, which can be determined using
routine laboratory techniques. Preparing expression constructs
encoding antigens and signal sequences is further described in, for
example, U.S. Pat. No. 6,500,641. Methods of secreting
non-secretable proteins are further described in, for example, U.S.
Pat. No. 6,472,176, and International Patent Application
Publication WO 02/48377.
[0043] The antigen encoded by the nucleic acid sequence of the gene
transfer vector also can be modified to attach or incorporate the
antigen on the host cell surface. In this respect, the antigen can
comprise a membrane anchor, such as a gpi-anchor, for conjugation
onto the cell surface. A transmembrane domain can be fused to the
antigen to incorporate a terminus of the antigen protein into the
cell membrane. Other strategies for displaying peptides on a cell
surface are known in the art and are appropriate for use in the
context of the invention.
[0044] It is believed that a portion of the shaft region of a
subgroup C adenovirus fiber protein provides a mechanism by which
adenovirus binds to and enters specific cell types, such as, for
example, dendritic cells and tumor cells. Thus, the inventive gene
transfer vector further comprises an amino acid sequence comprising
at least three (e.g., three or more, five or more, ten or more,
twenty or more, thirty or more, forty or more, or even fifty or
more) contiguous amino acids of a shaft region of a subgroup C
adenovirus fiber protein. The fiber protein of adenovirus is a
trimer (Devaux et al., J. Molec. Biol., 215, 567-588 (1990))
consisting of a tail, a shaft, and a knob. The fiber shaft region
is composed of repeating 15 amino acid motifs, which are believed
to form two alternating .beta.-strands and .beta.-bends (Green et
al., EMBO J., 2, 1357-1365 (1983), and Chroboczek et al., In P. B.
W. Doerfler, ed., The Molecular Repertoire of Adenoviruses, Vol. 1,
Springer-Verlag, Berlin, Germany, pp.163-200 (1995)). The overall
length of the fiber shaft region and the number of 15 amino-acid
repeats differ between adenoviral serotypes. For example, the Ad2
fiber shaft is 37 nanometers long and contains 22 repeats, whereas
the Ad3 fiber is 11 nanometers long and contains 6 repeats. A
receptor binding domain of the fiber protein is localized in the
knob region encoded by the last 200 amino acids of the protein
(Henry et al., J. Virology, 68(8), 5239-5246 (1994)). The regions
necessary for trimerization are also located in the knob region of
the protein (Henry et al. (1994), supra). A deletion mutant lacking
the last 40 amino acids of the knob region of the fiber protein
does not trimerize and also does not bind to penton base (Novelli
et al., Virology, 185, 365-376 (1991)). Thus, trimerization of the
fiber protein is necessary for penton base binding to the fiber.
Nuclear localization signals that direct the protein to the nucleus
to form viral particles following its synthesis in the cytoplasm
are located in the N-terminal region of the protein (Novelli et al.
(1991), supra). The fiber, together with the hexon, determine the
serotype specificity of the adenovirus (Watson et al., J. Gen.
Virol., 69, 525-535 (1988)).
[0045] The amino acid sequence comprising at least three contiguous
amino acids of a shaft region preferably is derived from a subgroup
C adenovirus fiber protein. In this regard, the amino acid sequence
can be derived from any suitable subgroup C adenovirus serotype.
Suitable subgroup C adenovirus serotypes include serotypes 1, 2, 5,
and 6. The amino acid sequence preferably comprises at least three
(e.g., 3, 10, 20, 30, 40, 50, or more) contiguous amino acids of a
shaft region of a serotype 2 or serotype 5 adenovirus fiber
protein. The amino acid sequence can be the entire shaft region of
a subgroup C (especially a serotype 2 or serotype 5) adenovirus
fiber protein. The amino acid sequence preferably comprises from
about 3 to about 50 contiguous amino acids (e.g., 15, 20, 30, 40,
or 50 amino acids) of a shaft region of a subgroup C (especially a
serotype 2 or serotype 5) adenovirus fiber protein. The amino acid
sequence more preferably comprises from about 3 to about 20
contiguous amino acids (e.g., about 5, 10, 15, or 20 amino acids)
of a shaft region of a subgroup C (especially a serotype 2 or
serotype 5) adenovirus fiber protein. Most preferably, the amino
acid sequence comprises the amino acid residues
lysine-lysine-threonine-lysine (KKTK) (SEQ ID NO: 1). For example,
the amino acid sequence can consist of the sequence KKTK.
Alternatively, SEQ ID NO: 1 can be part of a larger amino acid
sequence that is included in the inventive gene transfer vector. In
another embodiment, the amino acid sequence can comprise a fragment
of SEQ ID NO: 1, such as, for example, KKT (SEQ ID NO: 2) or KTK
(SEQ ID NO:3). The KKTK motif in the shaft region of Ad5 is known
to bind glycosaminoglycans (GAGs) (e.g., heparin and heparan
sulfate proteoglycans) (see, e.g., Hileman et al., Bioessays, 20,
156-67 (1998), and Smith et al., Mol Ther., 5, 770-9 (2002)). GAGs
are long unbranched polysaccharides containing a repeating
disaccharide unit. The disaccharide units contain either of two
modified sugars. The majority of GAGs in a human are linked to core
proteins, forming proteoglycans (also called mucopolysaccharides).
GAGs are located primarily on the surface of cells or in the
extracellular matrix (ECM). The amino acid sequences of the fiber
protein of a serotype 2 adenovirus and a serotype 5 adenovirus are
disclosed in Chroboczek et al., Virology, 161, 549-54 (1987).
[0046] One of ordinary skill in the art will appreciate that the
immune response elicited by the gene transfer vector once
administered to the mammal (e.g., a human) will depend upon the
location of the amino acid sequence comprising at least three
contiguous amino acids of a shaft region of a subgroup C adenovirus
fiber protein within the gene transfer vector. In this respect, the
amino acid sequence can be present in any suitable location within
the gene transfer vector. In this regard, the amino acid sequence
can be located on the surface of the gene transfer vector to
maximize recognition by the host immune system. When the gene
transfer vector is a viral vector, for example, the amino acid
sequence preferably is located on or within the virus capsid, as a
result of modification of the capsid proteins. Such modifications
include, for example, generating chimeric capsid proteins which
comprise the amino acid sequence. Such chimeric capsid proteins can
be generated using routine molecular biology and recombinant DNA
techniques (see, e.g., Sambrook et al., supra). Alternatively, it
is also possible to covalently attach the amino acid sequence to a
capsid protein of a viral gene transfer vector by chemical
modification, such as by chemical cross-linkage. Chemical
cross-linkage can occur using available sulfhydral or amide groups
on the viral vector. Such groups also can be found or introduced
into the the amino acid sequence comprising at least three
contiguous amino acids of a shaft region of a subgroup C adenovirus
fiber protein. Examples of suitable cross-linking agents include,
for example, 4-azidobenzoic acid (3-sulfo-N-succinimidyl) ester
Sodium salt (Sulfo-HSAB),
1,4-bis[3-(2-pyridyldithio)propionamido]butane, and
bis[2-(N-succinimidyl-oxycarbonyloxy)ethyl]sulfone. Other methods
for chemically cross-linking proteins are known in the art.
[0047] The invention further provides a method of inducing an
immune response in a mammal comprising administering to the mammal
a conjugate comprising (a) at least one antigen which induces an
immune response in the mammal, and (b) an amino acid sequence
comprising at least three contiguous amino acids of a shaft region
of a subgroup C adenovirus fiber protein, wherein the amino acid
sequence is not an intact adenovirus. Descriptions of the antigen
and the amino acid sequence comprising at least three contiguous
amino acids of a subgroup C adenoviral fiber protein set forth
above in connection with other embodiments of the invention also
are applicable to those same aspects of the aforesaid inventive
method of inducing an immune response.
[0048] A "conjugate" is a molecule that is generated via coupling
of two or more separate molecules. The inventive conjugate
preferably comprises the antigen chemically coupled to the amino
acid sequence of the shaft region of a subgroup C adenovirus. The
antigen can be chemically coupled to the amino acid sequence by any
suitable chemical bond, and preferably is coupled to the amino acid
sequence via covalent bonds. Suitable chemical bonds are well known
in the art and include disulfide bonds, acid labile bonds,
photolabile bonds, peptidase labile bonds, thioether bonds, and
esterase labile bonds. Such conjugates can be produced using
routine molecular biology techniques, such as those described in
Sambrook et al., supra.
[0049] Alternatively, the conjugate can be a fusion protein
comprising the antigen and the amino acid sequence of the shaft
region of a subgroup C adenovirus. The fusion protein can be
generated using routine molecular biology techniques, such as
restriction enzyme or recombinational cloning techniques (see,
e.g., Gateway.TM. cloning system (Invitrogen) and U.S. Pat. Nos.
5,314,995 and 5,994,104). When the conjugate is a fusion protein,
the invention further provides a nucleic acid molecule encoding the
fusion protein. In this respect, "nucleic acid molecule" is
intended to encompass a polymer of DNA or RNA, i.e., a
polynucleotide, which can be single-stranded or double-stranded and
which can contain non-natural or altered nucleotides. In one
embodiment, the nucleic acid molecule can lack introns or portions
thereof. The nucleic acid molecule preferably is DNA. The nucleic
acid molecule may be isolated or purified from any suitable source.
The nucleic acid molecule also may be chemically synthesized by
methods known in the art.
[0050] When the conjugate is a fusion protein, the fusion protein
also can include additional peptide sequences which act to promote
stability, purification, and/or detection of the fusion protein.
For example, a reporter peptide portion (e.g., green fluorescent
protein (GFP), .beta.-galactosidase, or a detectable domain
thereof) can be incorporated in the fusion protein.
Purification-facilitating peptide sequences include those derived
or obtained from maltose binding protein (MBP),
glutathione-S-transferase (GST), or thioredoxin (TRX). The fusion
protein also or alternatively can be tagged with an epitope which
can be antibody purified (e.g., the Flag epitope, which is
commercially available from Kodak (New Haven, Conn.)), a
hexa-histidine peptide, such as the tag provided in a pQE vector
available from QIAGEN, Inc. (Chatsworth, Calif.), or an HA tag (as
described in, e.g., Wilson et al., Cell, 37, 767 (1984)).
[0051] In the methods of the invention, the gene transfer vector or
conjugate preferably is administered to a mammal (e.g., a human),
wherein the antigen induces an immune response against the antigen.
The immune response can be a humoral immune response, a
cell-mediated immune response, or, desirably, a combination of
humoral and cell-mediated immunity. Ideally, the immune response
provides protection upon subsequent challenge with the antigen.
However, protective immunity is not required in the context of the
invention. The inventive method further can be used for antibody
production and harvesting.
[0052] To enhance the immune response generated against the
antigen, the gene transfer vector can further comprise a nucleic
acid sequence that encodes an immune stimulator, such as a
cytokine, a chemokine, or a chaperone. Cytokines include, for
example, Macrophage Colony Stimulating Factor (e.g., GM-CSF),
Interferon Alpha (IFN-.alpha.), Interferon Beta (IFN-.beta.),
Interferon Gamma (IFN-.gamma.), interleukins (IL-1, IL-2, IL-4,
IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-16, and IL-18),
the TNF family of proteins, Intercellular Adhesion Molecule-1
(ICAM-1), Lymphocyte Function-Associated antigen-3 (LFA-3), B7-1,
B7-2, FMS-related tyrosine kinase 3 ligand, (Flt3L), vasoactive
intestinal peptide (VIP), and CD40 ligand. Chemokines include, for
example, B Cell-Attracting chemokine-1 (BCA-1), Fractalkine,
Melanoma Growth Stimulatory Activity protein (MGSA), Hemofiltrate
CC chemokine 1 (HCC-1), Interleukin 8 (IL8), Interferon-stimulated
T-cell alpha chemoattractant (1-TAC), Lymphotactin, Monocyte
Chemotactic Protein 1 (MCP-1), Monocyte Chemotactic Protein 3
(MCP-3), Monocyte Chemotactic Protein 4 (MCP-4), Macrophage-Derived
Chemokine (MDC), a macrophage inflammatory protein (MIP), Platelet
Factor 4 (PF4), Regulated upon Activation, Normal T-cell Expressed,
and presumably Secreted (RANTES), breast and kidney cell chemokine
(BRAK), eotaxin, exodus 1-3, and the like. Chaperones include, for
example, the heat shock proteins Hsp170, Hsc70, and Hsp40.
Cytokines and chemokines are generally described in the art,
including the Invivogen catalog (2002), San Diego, Calif. Likewise,
the inventive conjugate can comprise any one or more of the
aforementioned immune stimulators.
[0053] Multiple gene transfer vectors can be administered to the
mammal, each gene transfer vector comprising one or more nucleic
acid sequences encoding one or more antigens and/or
immunomodulators. Similarly, multiple conjugates can be
administered to the mammal, each conjugate comprising one or more
antigens and/or immunomodulators. If the gene transfer vector
comprises more than one antigen-encoding nucleic acid sequence, two
or more nucleic acid sequences can be operably linked to the same
promoter (e.g., to form a bicistronic sequence), two or more
nucleic acid sequences can be operably linked to separate promoters
of the same type (e.g., the CMV promoter), or two or more nucleic
acid sequences can be operably linked to separate and different
promoters (e.g., the CMV promoter and .beta.-actin promoter). The
multiple gene transfer vectors can include two or more gene
transfer vector constructs encoding different antigens, different
epitopes of the same antigenic protein, the same antigenic protein
derived from different species or clades of microorganism, antigens
from different microorganisms, and the like. Similarly, the
multiple conjugates also can include two or more conjugates
comprising different antigens, different epitopes of the same
antigenic protein, the same antigenic protein derived from
different species or clades of microorganism, antigens from
different microorganisms, and the like. In will be appreciated
that, in some embodiments, administering a "cocktail" of gene
transfer vectors and/or conjugates having different antigens or
different epitopes of the same antigen can provide a more effective
immune response than administering a single gene transfer vector
clone or a single conjugate to a mammal.
[0054] Likewise, administering the gene transfer vector or the
conjugate can be one component of a multistep regimen for inducing
an immune response in a mammal. In particular, the inventive method
can represent one arm of a prime and boost immunization regimen.
The inventive method, for example, can comprise administering to
the mammal a priming gene transfer vector comprising a nucleic acid
sequence encoding at least one antigen prior to administering the
inventive gene transfer vector. The antigen of the priming gene
transfer vector or conjugate can be the same or different from the
antigen of the subsequently administered gene transfer vector or
conjugate. The subsequently administered gene transfer vector or
conjugate is then administered to boost the immune response to a
given pathogen. More than one boosting composition comprising the
gene transfer vector or conjugate can be provided in any suitable
timeframe (e.g., at least about 1 week, 2 weeks, 4 weeks, 8 weeks,
12 weeks, 16 weeks, or more following priming) to maintain
immunity.
[0055] The priming gene transfer vector or conjugate need not be a
gene transfer vector or conjugate as otherwise described herein for
use in the inventive methods. For example, any gene transfer vector
can be employed as a priming gene transfer vector, including, but
not limited to, a plasmid, a retrovirus, an adeno-associated virus,
a vaccinia virus, a herpesvirus, or a bacteriophage. Ideally, the
priming gene transfer vector is a plasmid. Alternatively, an immune
response can be primed or boosted by administration of the antigen
itself, e.g., an antigenic protein, inactivated pathogen, and the
like.
[0056] Any route of administration can be used to deliver the gene
transfer vector or the conjugate to the mammal. Indeed, although
more than one route can be used to administer the gene transfer
vector or the conjugate, a particular route can provide a more
immediate and more effective reaction than another route.
Preferably, the gene transfer vector or the conjugate is
administered via intramuscular injection. A dose of gene transfer
vector or conjugate also can be applied or instilled into body
cavities, absorbed through the skin (e.g., via a transdermal
patch), inhaled, ingested, topically applied to tissue, or
administered parenterally via, for instance, intravenous,
peritoneal, or intraarterial administration.
[0057] The gene transfer vector or conjugate can be administered in
or on a device that allows controlled or sustained release, such as
a sponge, biocompatible meshwork, mechanical reservoir, or
mechanical implant. Implants (see, e.g., U.S. Pat. No. 5,443,505),
devices (see, e.g., U.S. Pat. No. 4,863,457), such as an
implantable device, e.g., a mechanical reservoir or an implant or a
device comprised of a polymeric composition, are particularly
useful for administration of the gene transfer vector or conjugate.
The gene transfer vector or conjugate also can be administered in
the form of sustained-release formulations (see, e.g., U.S. Pat.
No. 5,378,475) comprising, for example, gel foam, hyaluronic acid,
gelatin, chondroitin sulfate, a polyphosphoester, such as
bis-2-hydroxyethyl-terephthalate (BHET), and/or a
polylactic-glycolic acid.
[0058] The dose of gene transfer vector or conjugate administered
to the mammal will depend on a number of factors, including the
type of gene transfer vector (e.g. virus or liposome) or conjugate
(e.g., fusion protein), the size of a target tissue, the extent of
any side-effects, the particular route of administration, and the
like. The dose ideally comprises an "effective amount" of gene
transfer vector or conjugate, i.e., a dose of gene transfer vector
or conjugate which provokes a desired immune response in the
mammal. The desired immune response can entail production of
antibodies, protection upon subsequent challenge, immune tolerance,
immune cell activation, and the like.
[0059] The gene transfer vector or conjugate desirably is
administered in a composition, preferably a physiologically
acceptable (e.g., pharmaceutically acceptable) composition, which
comprises a carrier, preferably a physiologically (e.g.,
pharmaceutically) acceptable carrier and the gene transfer
vector(s) or conjugate(s). Any suitable carrier can be used within
the context of the invention, and such carriers are well known in
the art. The choice of carrier will be determined, in part, by the
particular site to which the composition is to be administered and
the particular method used to administer the composition. The
composition can optionally be sterile or sterile with the exception
of the inventive gene transfer vector or conjugate.
[0060] Suitable carriers and their formulations are described in A.
R. Gennaro, ed., Remington: The Science and Practice of Pharmacy
(19th ed.), Mack Publishing Company, Easton, Pa. (1995).
Pharmaceutical carriers include sterile water, saline, Ringer's
solution, dextrose solution, and buffered solutions at
physiological pH. Typically, an appropriate amount of a
pharmaceutically acceptable salt is used in the formulation to
render the formulation isotonic. The pH of the formulation is
preferably from about 5 to about 8 (e.g., about 5.5, about 6, about
6.5, about 7, about 7.5, and ranges thereof). More preferably, the
pH is about 7 to about 7.5. Further carriers include
sustained-release preparations, such as semipermeable matrices of
solid hydrophobic polymers containing the gene transfer vector or
the conjugate, which matrices are in the form of shaped articles
(e.g., films, liposomes, or microparticles). It will be apparent to
those persons skilled in the art that certain carriers may be more
preferable depending upon, for instance, the route of
administration and concentration of composition being
administered.
[0061] Compositions (e.g., pharmaceutical compositions) comprising
the gene transfer vector or the conjugate can include carriers,
thickeners, diluents, buffers, preservatives, surface active
agents, and the like. The compositions can also include one or more
active ingredients, such as antimicrobial agents, anti-inflammatory
agents, anesthetics, and the like.
[0062] The composition comprising the gene transfer vector or
conjugate can be administered in any suitable manner depending on
whether local or systemic treatment is desired, and on the area to
be treated. Administration can be topical (including ophthalmical,
vaginal, rectal, transdermal, and the like), oral, by inhalation,
or parenteral (including by intravenous drip or subcutaneous,
intracavity, intraperitoneal, or intramuscular injection).
Inhalation administration refers to the delivery of the
compositions into the nose and nasal passages through one or both
of the nares and can comprise delivery by a spraying mechanism or
droplet mechanism, or through aerosolization of the nucleic acid,
vector, or fusion protein. Delivery can also be directly to any
area of the respiratory system (e.g., lungs) via intubation.
[0063] If the composition is to be administered parenterally, the
administration is generally by injection. Injectables can be
prepared in conventional forms, either as liquid solutions or
suspensions, solid forms suitable for suspension in liquid prior to
injection, or as emulsions. Additionally, parental administration
can involve the preparation of a slow-release or sustained-release
system, such that a constant dosage is maintained. Preparations for
parenteral administration include sterile aqueous or non-aqueous
solutions, suspensions, and emulsions. Examples of non-aqueous
solvents are propylene glycol, polyethylene glycol, vegetable oils,
such as olive oil, and injectable organic esters, such as ethyl
oleate. Aqueous carriers include water, alcoholic/aqueous
solutions, emulsions or suspensions, including saline and buffered
media. Parenteral vehicles include sodium chloride solution,
Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's,
or fixed oils. Intravenous vehicles include fluid and nutrient
replenishers, electrolyte replenishers (such as those based on
Ringer's dextrose), and the like. Preservatives and other additives
also can be present such as, for example, antimicrobials,
anti-oxidants, chelating agents, inert gases and the like.
[0064] Formulations for topical administration may include
ointments, lotions, creams, gels, drops, suppositories, sprays,
liquids, and powders. Conventional pharmaceutical carriers,
aqueous, powder, or oily bases, thickeners, and the like may be
necessary or desirable.
[0065] Compositions for oral administration include powders or
granules, suspensions or solutions in water or non-aqueous media,
capsules, sachets, or tablets. Thickeners, flavorings, diluents,
emulsifiers, dispersing aids, or binders may be desirable.
[0066] Some of the compositions can potentially be administered as
a pharmaceutically acceptable acid- or base-addition salt, formed
by reaction with inorganic acids, such as hydrochloric acid,
hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,
sulfuric acid, and phosphoric acid, and organic acids such as
formic acid, acetic acid, propionic acid, glycolic acid, lactic
acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,
maleic acid, and fumaric acid, or by reaction with an inorganic
base, such as sodium hydroxide, ammonium hydroxide, potassium
hydroxide, and organic bases, such as mono-, di-, trialkyl, and
aryl amines and substituted ethanolamines.
[0067] The gene transfer vector or conjugate can be administered
with a pharmaceutically acceptable carrier and can be delivered to
the mammal's cells in vivo and/or ex vivo by a variety of
mechanisms well-known in the art (e.g., uptake of naked DNA,
liposome fusion, intramuscular injection of DNA via a gene gun,
endocytosis, and the like).
[0068] If ex vivo methods are employed, cells or tissues can be
removed and maintained outside the body according to standard
protocols known in the art. The compositions can be introduced into
the cells via any gene transfer mechanism, such as calcium
phosphate mediated gene delivery, electroporation, microinjection,
or proteoliposomes. The transduced cells then can be infused (e.g.,
with a pharmaceutically acceptable carrier) or homotopically
transplanted back into the mammal per standard methods for the cell
or tissue type. Standard methods are known for transplantation or
infusion of various cells into a mammal.
[0069] In addition, one of ordinary skill in the art will
appreciate that the gene transfer vector or the conjugate can be
present in a composition with other therapeutic or
biologically-active agents. For example, factors that control
inflammation, such as ibuprofen or steroids, can be part of the
composition to reduce swelling and inflammation associated with in
vivo administration of the gene transfer vector or the conjugate.
As discussed herein, immune system stimulators can be administered
to enhance any immune response to the antigen. Antibiotics, i.e.,
microbicides and fungicides, can be present to treat existing
infection and/or reduce the risk of future infection, such as
infection associated with gene transfer procedures.
[0070] The invention further provides a gene transfer vector
comprising (a) a nucleic acid sequence encoding a protein, and (b)
an amino acid sequence comprising at least three contiguous amino
acids of a shaft region of a subgroup C adenovirus fiber protein,
wherein the gene transfer vector is not an adenoviral vector.
Descriptions of the gene transfer vector and the amino acid
sequence comprising at least three contiguous amino acids of a
subgroup C adenoviral fiber protein set forth above in connection
with other embodiments of the invention also are applicable to
those same aspects of the aforesaid inventive gene transfer vector.
The gene transfer vector can by any suitable gene transfer vector
set forth above (e.g., a viral vector, a liposome, or a virus-like
particle). The gene transfer vector preferably is a viral vector,
but is not an adenoviral vector.
[0071] The nucleic acid sequence encoding the protein can be
obtained from any source, e.g., isolated from nature, synthetically
generated, isolated from a genetically engineered organism, and the
like. An ordinarily skilled artisan will appreciate that any type
of nucleic acid sequence (e.g., DNA, RNA, and cDNA) that can be
inserted into a gene transfer vector can be used in connection with
the invention.
[0072] The nucleic acid sequence of the inventive gene transfer
vector preferably encodes a protein that is toxic to mammalian
(e.g., human) cells (i.e., a "cytotoxic" protein). Suitable toxic
proteins include, bacterial toxins, viral toxins, fungal toxins, or
toxins produced by a parasitic agent, plant toxins, cytotoxic drugs
(e.g., chemotherapeutic drugs), and radionuclides. Suitable
bacterial toxins include, for example, Aeromonas hydrophila
aerolysin toxin, Escherichia coli hemolysin toxin, the
enterotoxins, exfoliative toxins, toxic-shock toxins, and
.alpha.-toxin of Staphyloccocus aureus, Streptococcus pyogenes
streptolysin O toxin and pyrogenic exotoxins, diphtheria toxin,
Bacillus anthracis edema factor and lethal factor, Bordetella
pertussis dermonecrotic toxin and pertussis toxin, cholera toxin,
tetanus toxin, and Psuedomonas aeruginosa exotoxin A. Suitable
viral toxins include, for example, herpes simplex virus thymidine
kinase (TK), and any toxin produced by a virus from the family
Hepadnaviridae, Parvoviridae (e.g., adeno-associated viruses
(AAVs)), Papovaviridae (e.g., the papillomaviruses), Adenoviridae
(e.g., human adenovirus), Picornaviridae (e.g., hepatitis A virus),
Herpesviridae (e.g., the herpes simplex-like viruses), Retroviridae
(e.g., HIV), or any other virus family described herein. The
protein encoded by the nucleic acid sequence of the inventive gene
transfer vector, however, is not limited to these exemplary
proteins. Indeed, any protein that is cytotoxic to a mammalian
cell, preferably a human cell is within the scope of the
invention.
[0073] In another embodiment, the nucleic acid sequence of the
inventive gene transfer vector can encode an antigen. In this
respect, the nucleic acid sequence can encode any suitable antigen,
such as those described herein.
[0074] The nucleic acid sequence of the inventive gene transfer
vector preferably encodes a secreted protein, e.g., a protein that
is naturally secreted by the infected cell. By "secreted protein"
is meant any peptide, polypeptide, or portion thereof, which is
released by a cell into the extracellular environment. The nucleic
acid sequence also can encode a protein that is not naturally
secreted by the cell, but which is released by cell lysis induced
by gene transfer vector (e.g., viral vector) transduction.
Alternatively, the nucleic acid sequence can encode a protein that
is not naturally secreted by the cell (i.e., a non-secretable
protein), but which comprises a signal peptide that facilitates
protein secretion (see, e.g., U.S. Pat. No. 6,472,176). In this
manner, for example, the nucleic acid sequence encodes an
endoplasmic reticulum (ER) localization signal peptide and the
non-secretable protein. The ER localization signal peptide
functions to direct DNA, RNA, and/or a protein to the membrane of
the endoplasmic reticulum, wherein a protein is expressed and
targeted for secretion. The ER localization signal peptide
desirably functions to increase the secretion (i.e., the secretion
potential) by a cell of (i) proteins that are not normally secreted
(i.e., secretable) by the cell and/or (ii) proteins that are
normally secreted by a cell, but in low (i.e., less than desired)
quantities. The ER localization signal peptide encoded by the
polynucleotide can be any suitable ER localization signal peptide
or polypeptide (i.e., protein). For example, the ER localization
signal peptide encoded by the nucleic acid sequence can be a
peptide or polypeptide (i.e., protein) selected from the group
consisting of nerve growth factor (NGF), immunoglobulin (Ig) (e.g.,
an Ig .kappa. chain leader sequence), and midkine (MK), or a
portion thereof. Suitable ER localization signal peptides also
include those described in Ladunga, Current Opinions in
Biotechnology, 11, 13-18 (2000).
[0075] The nucleic acid sequence can encode a tumor necrosis factor
(TNF), a vascular endothelial growth factor (VEGF), or a pigment
epithelium-derived factor (PEDF). Preferably, the gene transfer
vector comprises a nucleic acid sequence coding for a TNF. Nucleic
acid sequences encoding a TNF include nucleic acid sequences
encoding any member of the TNF family of proteins (e.g., CD40
ligand and Fas ligand). The gene transfer vector preferably
comprises a nucleic acid sequence encoding human TNF-.alpha.. A
nucleic acid sequence coding for TNF is described in U.S. Pat. No.
4,879,226. Alternatively, the nucleic acid sequence can encode a
VEGF. The nucleic acid sequence can encode any suitable VEGF
isoform, including, but not limited to, VEGF.sub.121, VEGF.sub.145,
VEGF.sub.165, VEGF.sub.189, or VEGF.sub.206, which are variously
described in U.S. Pat. Nos. 5,332,671, 5,240,848, and 5,219,739.
Most preferably, because of their higher biological activity, the
nucleic acid sequence encodes VEGF.sub.121 or VEGF.sub.165,
particularly VEGF.sub.121. A notable difference between
VEGF.sub.121 and VEGF.sub.165 is that VEGF.sub.121 does not bind to
heparin with a high degree of affinity, as does VEGF.sub.165. Other
suitable VEGF peptides are VEGF-II, VEGF-C, and the like. The
nucleic acid sequence also can encode a PEDF. PEDF, also known as
early population doubling factor-1 (EPC-1), is a secreted protein
having homology to a family of serine protease inhibitors named
serpins. PEDF is made predominantly by retinal pigment epithelial
cells and is detectable in most tissues and cell types of the body.
PEDF has both neurotrophic and anti-angiogenic properties and,
therefore, is useful in the treatment and study of a broad array of
diseases. Nucleic acid sequences encoding anti-angiogenic
derivatives of PEDF, known as SLED proteins (see, e.g.,
International Patent Application Publication No. WO 99/04806), also
can be used in connection with the invention. PEDF is further
characterized in International Patent Application Publication Nos.
WO 93/24529 and WO 99/04806, and the nucleic acid sequence encoding
PEDF is described in U.S. Pat. No. 5,840,686.
[0076] The inventive gene transfer vector can comprise one or more
additional nucleic acid sequences, each encoding one or more gene
products, such that one or more additional proteins are expressed
in a host cell. The expression of the additional gene product(s)
can be separately regulated by individual expression control
sequences, or coordinately regulated by one common expression
control sequence. Alternatively, the expression of the additional
nucleic acid(s) can be regulated by the same expression control
sequence that regulates expression of the previously described
nucleic acid sequence encoding the protein; however, any
transcription terminating regions present in the nucleic acid
encoding the protein would be eliminated to allow for
transcriptional read-through of the additional nucleic acid
sequence(s). The additional nucleic acid sequence(s) can comprise
any suitable expression control sequence(s) and any suitable
transcription-termination region(s) discussed herein in connection
with the previously described nucleic acid sequence encoding the
protein.
[0077] The nucleic acid sequence can encode any variant, homolog,
or functional portion of the aforementioned proteins. A variant of
the protein can include one or more mutations (e.g., point
mutations, deletions, insertions, etc.) from a corresponding
naturally occurring protein. By "naturally occurring" is meant that
the protein can be found in nature and has not been synthetically
modified. When mutations are introduced in the nucleic acid
sequence encoding the protein, such mutations desirably will effect
a substitution in the encoded protein whereby codons encoding
positively-charged residues (H, K, and R) are substituted with
codons encoding positively-charged residues, codons encoding
negatively-charged residues (D and E) are substituted with codons
encoding negatively-charged residues, codons encoding neutral polar
residues (C, G, N, Q, S, T, and Y) are substituted with codons
encoding neutral polar residues, and/or codons encoding neutral
non-polar residues (A, F, I, L, M, P, V, and W) are substituted
with codons encoding neutral non-polar residues. In addition, a
homolog of the protein can be any peptide, polypeptide, or portion
thereof, that is more than about 70% identical (preferably more
than about 80% identical, more preferably more than about 90%
identical, and most preferably more than about 95% identical) to
the protein at the amino acid level. The degree of amino acid
identity can be determined using any method known in the art, such
as the BLAST sequence database. Furthermore, a homolog of the
protein can be any peptide, polypeptide, or portion thereof, which
hybridizes to the protein under at least moderate, preferably high,
stringency conditions. Exemplary moderate stringency conditions
include overnight incubation at 370.degree. C. in a solution
comprising 20% formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium
citrate), 50 mM sodium phosphate (pH 7.6), 5.times. Denhardt's
solution, 10% dextran sulfate, and 20 mg/ml denatured sheared
salmon sperm DNA, followed by washing the filters in 1.times.SSC at
about 37-50.degree. C., or substantially similar conditions, e.g.,
the moderately stringent conditions described in Sambrook et al.,
supra. High stringency conditions are conditions that use, for
example (1) low ionic strength and high temperature for washing,
such as 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium
dodecyl sulfate (SDS) at 50.degree. C., (2) employ a denaturing
agent during hybridization, such as formamide, for example, 50%
(v/v) formamide with 0.1% bovine serum albumin (BSA)/0.1%
Ficoll/0.1% polyvinylpyrrolidone (PVP)/50 mM sodium phosphate
buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate
at 42.degree. C., or (3) employ 50% formamide, 5.times.SSC (0.75 M
NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8),
0.1% sodium pyrophosphate, 5.times. Denhardt's solution, sonicated
salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran sulfate
at 42.degree. C., with washes at (i) 42.degree. C. in
0.2.times.SSC, (ii) at 55.degree. C. in 50% formamide and (iii) at
55.degree. C. in 0.1.times.SSC (preferably in combination with
EDTA). Additional details and an explanation of stringency of
hybridization reactions are provided in, e.g., Ausubel et al.,
supra. A "functional portion" is any portion of a protein that
retains the biological activity of the naturally occurring,
full-length protein at measurable levels.
[0078] The expression of the nucleic acid sequence is controlled by
any suitable promoter. Ideally, the nucleic acid sequence is
operably linked to a promoter and a polyadenylation sequence as
described herein.
[0079] The invention also provides a conjugate comprising (a) a
protein or a non-proteinaceous molecule, and (b) an amino acid
sequence comprising at least three contiguous amino acids of a
shaft region of a subgroup C adenovirus fiber protein, wherein when
the conjugate comprises a protein, the conjugate is not an
adenovirus. Descriptions of the conjugate and the amino acid
sequence comprising at least three contiguous amino acids of a
shaft region of a subgroup C adenovirus fiber protein set forth
above in connection with other embodiments of the invention also
are applicable to those same aspects of the aforesaid inventive
conjugate. In this respect, the conjugate preferably comprises the
protein or non-proteinaceous molecule chemically coupled to the
amino acid sequence of the shaft region of a subgroup C adenovirus.
The protein or non-proteinaceous molecule can be chemically coupled
to the amino acid sequence by any suitable chemical bond, and
preferably is coupled to the amino acid sequence via covalent
bonds, such as those described herein. More preferably, the
inventive conjugate is a fusion protein.
[0080] When the conjugate comprises a protein, the conjugate
preferably is not an adenovirus, especially an encapsidated
adenovirus. An encapsidated adenovirus comprises an adenoviral
genome contained within an adenovirus capsid. An encapsidated
adenovirus can be replication-competent, conditionally
replication-deficient, or replication-deficient. A conditionally
replication-deficient adenovirus is engineered to replicate under
conditions pre-determined by the practitioner. A
replication-deficient adenovirus comprises an adenoviral genome
that lacks at least one replication-essential gene function (i.e.,
such that the adenoviral vector does not replicate in typical host
cells, especially those in a human patient that could be infected
by the adenoviral vector).
[0081] In one embodiment, the conjugate comprises one or more
proteins. The protein can be any suitable protein described herein.
Suitable proteins include, for example, an antigen, a cytotoxic
protein (e.g., a bacterial or viral toxin), a secreted protein
(e.g., a TNF, VEGF, or PEDF), and a protein that enhances immune
responses (e.g., TNF, GMCSF or IL-12).
[0082] In another embodiment, the conjugate comprises a
non-proteinaceous molecule. The conjugate can comprise any suitable
non-proteinaceous molecule. Suitable non-proteinaceous molecules
include, for example, carbohydrates, lipids, and non-proteinaceous
small molecules. By "small molecule" is meant any molecule that is
not considered a macromolecule, which includes a nucleic acid, a
carbohydrate, or a lipid. Small molecules are a diverse group of
natural and synthetic substances that generally have a low
molecular weight. For example, a small molecule typically has a
molecular weight of less than about 1,000 (e.g., less than about
700, 500, or 300). Small molecules can be isolated from natural
sources such as plants, fungi, or microbes, or they can be
synthesized by organic chemistry. Most conventional
pharmaceuticals, such as aspirin, penicillin, and
chemotherapeutics, are small molecules. The non-proteinaceous
molecule preferably is a hapten. A hapten is a low molecular weight
molecule which contains an antigenic determinant, but which itself
is not antigenic unless complexed with an antigenic carrier.
Examples of suitable haptens for use in the invention include
dinitrophenols, phosphrylcholine, and dextran.
[0083] The invention provides a method of delivering a protein or a
non-proteinaceous molecule to a cell. The method comprises
contacting the cell with a gene transfer or conjugate as described
herein.
[0084] The protein or non-proteinaceous molecule can be delivered
to any suitable cell. As previously mentioned, it is believed that
a portion of the shaft region of a subgroup C adenovirus fiber
protein is responsible for targeting adenovirus to specific cell
types (e.g., immune cells and tumor cells). In accordance with the
inventive method, the protein or non-proteinaceous molecule can be
delivered to immune cells, preferably antigen presenting cells,
such as dendritic cells, monocytes, and macrophages.
[0085] When dendritic cells are the desired target cell, the gene
transfer vector or the conjugate, by way of the amino acid sequence
of a subgroup C adenovirus fiber shaft region, recognizes a protein
typically found on dendritic cell surfaces such as adhesion
proteins, chemokine receptors, complement receptors, co-stimulation
proteins, cytokine receptors, high level antigen presenting
molecules, homing proteins, marker proteins, receptors for antigen
uptake, signaling proteins, virus receptors, etc. Examples of such
potential ligand-binding sites in dendritic cells include
.alpha..sub.v.beta..sub.3 integrins, .alpha..sub.v.beta..sub.5
integrins, 2A1, 7-TM receptors, CD1, CD11a, CD11b, CD11c, CD21,
CD24, CD32, CD4, CD40, CD44 variants, CD46, CD49d, CD50, CD54,
CD58, CD64, ASGPR, CD80, CD83, CD86, E-cadherin, integrins, M342,
MHC-I, MHC-II, MIDC-8, MMR, OX62, p200-MR6, p55, S100, TNF-R, etc.
When dendritic cells are targeted, the amino acid sequence
comprising at least three contiguous amino acids of a shaft region
of a subgroup C adenovirus fiber protein preferably recognizes the
CD40 cell surface protein.
[0086] When macrophages are the desired target, the gene transfer
vector or the conjugate, by way of the amino acid sequence of a
subgroup C adenovirus fiber shaft region, recognizes a protein
typically found on macrophage cell surfaces, such as
phosphatidylserine receptors, vitronectin receptors, integrins,
adhesion receptors, receptors involved in signal transduction
and/or inflammation, markers, receptors for induction of cytokines,
or receptors up-regulated upon challenge by pathogens, members of
the group B scavenger receptor cysteine-rich (SRCR) superfamily,
sialic acid binding receptors, members of the Fc receptor family,
B7-1 and B7-2 surface molecules, lymphocyte receptors, leukocyte
receptors, antigen presenting molecules, and the like. Examples of
suitable macrophage surface target proteins include, but are not
limited to, heparin sulfate proteoglycans,
.alpha..sub.v.beta..sub.3 integrins, .alpha..sub.v.beta..sub.5
integrins, B7-1, B7-2, CD11c, CD13, CD16, CD163, CD1a, CD22, CD23,
CD29,Cd32, CD33, CD36, CD44, CD45, CD49e, CD52, CD53, CD54, CD71,
CD87, CD9, CD98, Ig receptors, Fc receptor proteins (e.g., subtypes
of Fc.alpha., Fc.gamma., Fc.epsilon., etc.), folate receptor b, HLA
Class I, Sialoadhesin, siglec-5, and the toll-like receptor-2
(TLR2).
[0087] When B-cells are the desired target, the gene transfer
vector or the conjugate, by way of the amino acid sequence of a
subgroup C adenovirus fiber shaft region, recognizes a protein
typically found on B-cell surfaces, such as integrins and other
adhesion molecules, complement receptors, interleukin receptors,
phagocyte receptors, immunoglobulin receptors, activation markers,
transferrin receptors, members of the scavenger receptor
cysteine-rich (SRCR) superfamily, growth factor receptors,
selectins, MHC molecules, TNF-receptors, and TNF-R associated
factors. Examples of typical B-cell surface proteins include
.beta.-glycan, B cell antigen receptor (BAC), B7-2, B-cell receptor
(BCR), C3d receptor, CD1, CD18, CD19, CD20, CD21, CD22, CD23, CD35,
CD40, CD5, CD6, CD69, CD69, CD71, CD79a/CD79b dimer, CD95,
endoglin, Fas antigen, human Ig receptors, Fc receptor proteins
(e.g., subtypes of Fca, Fcg, Fc.epsilon., etc.), IgM, gp200-MR6,
Growth Hormone Receptor (GH-R), ICAM-1, ILT2, CD85, MHC class I and
II molecules, transforming growth factor receptor (TGF-R),
.alpha..sub.4.beta..sub.7 integrin, and .alpha..sub.v.beta..sub.3
integrin.
[0088] In accordance with the inventive method, the protein or
non-proteinaceous molecule can be delivered to a tumor cell. When
tumor cells are the desired target, the gene transfer vector or the
conjugate, by way of the amino acid sequence of a subgroup C
adenovirus fiber shaft region, recognizes a protein typically found
on tumor cell surfaces. The tumor cell can be associated with
cancers of (i.e., located in) the oral cavity and pharynx, the
digestive system, the respiratory system, bones and joints (e.g.,
bony metastases), soft tissue, the skin (e.g., melanoma), breast,
the genital system, the urinary system, the eye and orbit, the
brain and nervous system (e.g., glioma), or the endocrine system
(e.g., thyroid). The tumor cell can be associated with cancers of
the oral cavity including, for example, the tongue and tissues of
the mouth. The tumor cell can be associated with cancers of the
digestive system including, for example, the esophagus, stomach,
small intestine, colon, rectum, anus, liver, gall bladder, and
pancreas. The tumor cell can be associated with cancers of the
respiratory system, including, for example, the larynx, lung, and
bronchus (e.g., non-small cell lung carcinoma). The tumor cell can
be associated with cancers of the uterine cervix, uterine corpus,
ovary vulva, vagina, prostate, testis, and penis, which make up the
male and female genital systems, and the urinary bladder, kidney,
renal pelvis, and ureter, which comprise the urinary system. The
tumor cell also can be associated with lymphoma (e.g., Hodgkin's
disease and Non-Hodgkin's lymphoma), multiple myeloma, or leukemia
(e.g., acute lymphocytic leukemia, chronic lymphocytic leukemia,
acute myeloid leukemia, chronic myeloid leukemia, and the
like).
[0089] The gene transfer vector and/or the conjugate can contact a
particular cell in vivo or ex vivo using methods known in the art.
In this respect, the gene transfer vector and/or the conjugate can
be administered with a pharmaceutically acceptable carrier and can
be delivered to the mammal's cells, preferably human cells, in vivo
and/or ex vivo by a variety of mechanisms well-known in the art,
such as those described herein. The gene transfer vector and/or the
conjugate preferably contacts one or more cells in vivo.
[0090] The following examples further illustrate the invention but,
of course, should not be construed as in any way limiting its
scope.
EXAMPLE 1
[0091] This example demonstrates the ability of an adenovirus
ablated for native host cell binding to induce an immune response
in a mammal and to transduce immune cells.
[0092] A panel of adenovirus vectors was constructed to test the
contribution of the shaft region of the adenovirus serotype 5 (Ad5)
fiber protein to immunogenicity. Ad5 vectors that expressed the
green fluorescent protein (GFP) were modified in the penton base
and fiber proteins. Specifically, an integrin binding motif,
Arg-Gly-Asp (RGD) was deleted from the penton base protein, and an
amino acid residue of the knob region of the fiber protein that
mediates interaction of the fiber with the Cocksaxie B and
Adenovirus Receptor (CAR) also was deleted. This "double-ablated"
Ad5 vector was referred as Adf.DA.
[0093] An adenovirus serotype 35 (Ad35) vector was modified such
that the shaft region of the Ad35 fiber protein was replaced with
the shaft region of the Ad5 fiber protein using routine recombinant
DNA techniques. This Ad35 vector, referred to as Ad35f.5S, also
encoded the GFP protein. GFP-expressing, wild type capsid vectors
of adenovirus serotypes 35 and 5, referred to as Ad35f and Adf,
respectively, served as controls.
[0094] Groups of five Balb/c mice were immunized with the
above-described adenoviral vectors over a dose range of
1.times.10.sup.8 to 1.times.10.sup.10 particles (pu). The
percentage of T-cells that were immunoreactive to the GFP antigen
was determined by intra-cellular cytokine staining (see FIGS. 1A
and 1B). Animals immunized with Ad35f.5S exhibited the highest
percentage of cytokine positive CD8+ T cells, and exhibited the
same level of CD4+ T cells as the control Adf vector. Ad35f.5S
induced levels of CD8+ T cells that significantly exceeded the
levels induced by Ad35f. The animals immunized with AdfDA also
mounted a significant immune response to GFP, and the levels of
CD4+ and CD8+ T cells were at least equal to those animals
immunized with Adf.
[0095] The ability of the double-ablated vector, and the role of
the Ad5 shaft region, in transducing cells of the immune system
were determined. Murine bone marrow cells were cultured for 1 week
with GMCSF, infected with an E1-deficient Ad5 encoding a luciferase
reporter gene (AdL), AdLDA, or Adf in either the absence or
presence of inhibitors of interaction with heparan sulfate
proteoglycans. Competitors were heparin (Sigma Aldrich H6279) or
heparan (heparan sulfate proteoglycan, Sigma Aldrich H4777). The
population of cells was enriched for dendritic cells by sorting
with flow cytometry on CD19 and CD11c. This population of cells is
referred to as myeloid dendritic cells (see, e.g., Inaba et al., J
Exp. Med., 176, 1693-1702 (1992)). In the absence of competitor,
the luciferase activity of CD19-negative, CD11c-positive cells was
equivalent between populations infected by AdLDA or AdL (see FIG.
2, AdlucDA and Adluc, respectively). In the presence of competitor,
the level of luciferase activity from cells infected with AdLDA was
approximately 100-fold lower, showing the dependency of
transduction on interaction with heparan sulfate proteoglycan.
Since the KKTK motif in the shaft region of Ad5 is known to be a
heparan sulfate proteoglycan binding motif (see, e.g., Hileman et
al., supra, and Smith et al., supra), these results demonstrate
that the double-ablated vector transduces professional antigen
presenting cells through a fiber shaft dependent mechanism.
[0096] The double-ablated vector was next tested for transduction
of tumor cells. The double-ablated vector showed differential
transduction relative to the wild type capsid vector. AdL.DA
transduction of 293-ORF6 cells resulted in 1000-fold less
luciferase activity compared to AdL, whereas the luciferase
activity in Caov3 cells, a human adenocarcinoma cell line, was
equivalent between AdL and AdL.DA (see FIG. 3A). Similar results
were obtained with two murine cell lines (see FIG. 3B).
[0097] This example demonstrates that the immune response induced
by Ad5 and Ad35 vectors is independent of interactions mediated by
penton base protein and the CAR-binding region of fiber protein.
Additionally, this example demonstrates that the shaft region of
the Ad5 fiber confers specificity for transducing cells of the
immune system (e.g., myeloid dendritic cells), as well as tumor
cells (e.g., human ovarian adenocarcinoma).
EXAMPLE 2
[0098] This example demonstrates the preparation of the inventive
gene transfer vector.
[0099] An adeno-associated virus vector is prepared in accordance
with the methods disclosed in Warrington et al., J. Virol., 78,
6595-6609 (2004). In particular, an amino acid sequence comprising
the amino acid sequence KKTK of the shaft region of an adenovirus
serotype 5 fiber protein is inserted at amino acid position 138
within the overlap region of the AAV V1 and V2 capsid proteins. The
resulting AAV vector is further engineered to contain an antigen,
such as the GAG antigen of HIV. The AAV vector exhibits enhanced
immunogenicity when administered to a human as compared to an
unmodified AAV vector.
EXAMPLE 3
[0100] This example demonstrates the preparation of the inventive
conjugate.
[0101] An amino acid sequence comprising the amino acid sequence
KKTK (SEQ ID NO: 1) of the shaft region of an adenovirus serotype 5
fiber protein is conjugated to the N-terminus or C-terminus of a
recombinant anthrax protective antigen vaccine or a recombinant
anthrax lethal factor vaccine (as reviewed in, e.g., Leppla et al.,
J. Clin. Invest., 109, 141-144 (2002). The conjugate exhibits
enhanced immunogenicity when administered to a human as compared to
the unmodified protective antigen vaccine or the lethal factor
vaccine.
[0102] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0103] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0104] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
3 1 4 PRT Adenovirus 1 Lys Lys Thr Lys 1 2 3 PRT Adenovirus 2 Lys
Lys Thr 1 3 3 PRT Adenovirus 3 Lys Thr Lys 1
* * * * *